Our goal is to study gene regulation in Alzheimer's disease (AD), based on the "amyloid hypothesis" of Alzheimer's disease. Overproduction of the amyloid beta-peptide (Abeta) causes a cascade of neurodegenerative steps resulting in plaque formation and neuronal loss that characterize Alzheimer's disease. The unresolved key question in the field is what factors cause overproduction of Abeta and its large Abeta precursor protein (APP). Increased Abeta production may result from an increase in APP expression, or in its proteolytic processing by the limiting beta-APP cleaving enzyme (BACE). The goals of this proposal are to investigate the transcriptional regulation of i) APP, because APP (and, hence, AP) biogenesis begins at the level of transcription, and ii) BACE gene, as Abeta overproduction may be due to increased BACE level as a result of upregulation in this gene. Specific Aims are: 1) To study the functional domains of the APP promoter and effects of different agents on its activity. We will functionally characterize the 7.9 kb APP promoter and study how intrinsic (cytokines) and extrinsic (metals) factors regulate promoter activity. Promoter will be studied by serial deletions, mutagenesis and transfection experiments in different cell types and primary neuronal cultures. 2) To identify the effects of specific factors and cvtokines common to both APP and BACE gene regulation. We will characterize the role of IL-1alpha, TNF-alpha and CREB transcription factor (TF) on 4.1kb BACE promoter activity. 3) To identify cell type-specific nuclear factors. A 30 bp novel region (-76-47) of the APP promoter contains a regulatory domain that interacts with at least two proteins, PuF and SkiP. We will test i) the candidate TFs that control APP promoter activity and ii) the status of such TF in normal and AD brain tissues using gel shift assay and DNA-affinity chromatography. 4) To characterize APP gene polymorphisms that influences the risk of late-onset Alzheimer's disease. We discovered two polymorphisms at -3829 and -1023 that may be associated with Alzheimer's disease. We will i) do functional and DNA-protein binding studies with promoter variants and ii) correlate promoter studies with levels of APP and Abeta. 5) To study the APP-5'-UTR region. APP expression is also regulated via the 5'-untranslated region (UTR). We will test a dual role for the APP5'-UTR at both transcriptional and post-transcriptional levels, and study its interaction with cytokines. Cell lines from families with characterized FAD will be analyzed for differential expression of the APP and BACE genes. Studying APP and BACE gene regulation is crucial to understand APP production leading to Aa generation. These studies should help developing suitable drug targets for the treatment of Alzheimer's disease.